A carbon paste electrode modified with ZnO nanoparticle (ZnO/NPs) and room-temperature ionic liquid, 1-methyl-3-butylimidazolium chloride was fabricated. The direct electro-oxidation behavior of acetaminophen (AC) was carefully studied by cyclic voltammetry (CV), chronoamperometry and electrochemical impedance spectroscopy (EIS) and the kinetic parameters such as the electron transfer coefficient, α, and the diffusion coefficient, D of AC at the IL/ZnO/NPs/CPE surface was estimated. The results exhibited remarkable increase in the electron transfer rate and significant decrease in the overpotential for AC oxidation reaction in contrast to that on the bare carbon paste electrode (CPE), also, the sensor effectively resolved the overlapping anodic peaks of AC and dihydronicotinamide adenine dinucleotide (NADH). Under the optimal conditions, the oxidation peak current was linear to the AC concentration over the range of 0.1 to 550 μmol L−1 with a detection limit of 0.07μmol L−1. The proposed sensor has been successfully applied in urine, serum and pharmaceutical samples with satisfactory results.